In the lumen of the colon, short chain fatty acids (SCFAs) such as acetate, propionate and butyrate are the predominant anions. SCFAs stimulate colonic electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes, and SCFAs also provide metabolic support for colonocytes. Lowering of luminal SCFA availability has been associated with the pathogenesis of various colitides, and rectal irrigation with SCFAs is currently being appraised as a potential treatment for ulcerative colitis. The physiologic and pathophysiologic roles for SCFAS imply that we need a more complete understanding of the mechanisms of action of these compounds. The long-term objective of this work is to define mechanisms by which SCFAs regulate Na/H exchange in colonocytes, because recent evidence shows that the currently accepted model for this process is insufficient to describe experimental observations. Two model systems will be used: mouse distal colonic epithelium and HT29-C1 cells. In both models, preliminary results suggest that newly identified microdomains of pH within the epithelium will contribute to selective activation of polarized Na/H exchangers by SCFAs. A unique aspect of the proposal is the application of quantitative confocal microscopy to evaluate epithelial function along the crypt-to-surface axis of the colon or along the basal- to-apical axis of individual colonocytes; while maintaining intact epithelial architecture and independently superfusing the apical and basolateral surfaces of epithelia. One specific aim will determine if SCFAs regulate colonocyte Na/H exchange via changes in pH. Experimental methods to be used include isotopic fluxes and optical (confocal or digital imaging microscopy) measurements of intracellular or extracellular pH using SNARF-1 (a pH-sensitive fluorescent dye). Experiments are designed to determine all pH microdomains which are regulated by SCFAs, and to define sites in the epithelium at which SCFAs impose selective activation of polarized Na/H exchangers. It will be tested if changes in pH are necessary and sufficient to explain how transepithelial SCFA gradients impose selective activation of polarized Na/H exchangers. A second aim will define which membrane transporters are responsible for the ability of SCFAs to stimulate apical Na/H exchange. Experiments define mechanisms mediating and regulating SCFA flux across the apical and basolateral membranes of mouse colonocytes, and establish distinguishing kinetic characteristics (and ideally the molecular identity) of colonic Na/H exchangers activated by SCFAs. The outcome of these experiments will help resolve current controversy about the mechanisms of SCFA transport in colonic epithelium, and the mechanism of SCFA-stimulated sodium absorption. It is hoped that this will contribute to better understanding of SCFA action in health and colonic disease.